In 1983, a retrovirus, known as human immunodeficiency virus type 1 (HIV-1), was established as a causitive agent of acquired immune deficiency syndrome (AIDS), see R. C. Gallo and L. Montagnier, Scientific American, 259 (4), 40 (1988). This virus has become a pestilence of alarming proportion. More recently, the closely related virus, human immunodeficiency virus type 2 (HIV-2) has been identified as a second causative agent of AIDS.
The identification of human immunodeficiency virus (HIV) as a causative agent and the development of methods to grow the virus in quantity have resulted in the discovery of compounds which inhibit the replication of HIV in vitro. The most important class of inhibitor compounds identified in this manner is a group of dideoxynucleosides of which 3'-azido-3'-deoxythymidine (known also as zidovudine or AZT) is used therapeutically to manage certain patients with symptomatic HIV infections. This class of compounds has been found to interfere with the life cycle of HIV by inhibiting reverse transcriptase. This enzyme converts viral RNA to double-stranded deoxyribonucleic acid (DNA) and as such is an essential enzyme for HIV replication. In addition to inhibiting reverse transcriptase, other stages of the HIV life cycle have been identified as targets for developing anti-AIDS drugs. One target that is receiving increased attention is an HIV-encoded enzyme known as HIV protease. This enzyme, like the reverse transcriptase, is encoded by the gag-pol gene and is essential for HIV growth. It is responsible for effecting certain cleavages within the gag (p55) or gag-pol (p180) protein precursors to release structural proteins and enzymes, including itself, found in mature infectious virions. Soon after infection, the protease may cleave the core nucleocapsid, thus triggering conformational changes of the ribonucleoprotein substrate and activating DNA synthesis. Thus, inhibitors of HIV protease may block several stages in the HIV life cycle. For a recent review on HIV-protease inhibitors, see B. M. Dunn and J. Kay, Antiviral Chemistry & Chemotherapy, 1, 3, (1990).
Notwithstanding the progress that has been made in the causes and treatment of AIDS, better small animal models are needed to study HIV infections and to evaluate potential drugs and vaccines. The need has resulted in the development of two small animal models based on severe combined immunodeficient (SCID) mice, D. E. Mosier et al., Nature, 335, 256 (1988) and J. M. McCune et al., Science, 241, 1632 (1988); These small animal models have the potential of being used for evaluating anti-AIDS drugs and vaccines. However, there is still a need for a small animal model to study HIV/host cell interactions and to screen for anti-HIV drugs.
The present invention offers to fulfil the latter need by providing a transgenic non-human mammal, for example a mouse or a rat, which expresses non-infectious HIV RNA, and the structural proteins, regulatory proteins and enzymes translated thereform, and which can heritably transmit the transgene to its progeny.
The procedure for producing a transgenic animal is known in the art; for example, see B. Hogan et al., "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA, 1986 and T. E. Wagner and P. C. Hoppe, U.S. Pat. No. 4,873,191, issued Oct. 10, 1989. However, the prior art also teaches that it is difficult, and not precisely understood how, to obtain an animal which carries a transgene functioning in a suitable physiological environment and in a desired manner, and which can produce offspring expressing the gene. The desired gene expression can be nullified if the cloned DNA is integrated into a region of the animal's chromosome that modifies its expression, or if it undergoes mutation or rearrangement in the process of being integrated into the chromosome; see, for example, H. Van der Putten et al., Mol. Gen. Genetic, 198, 128 (1984). Accordingly, a significant advance in the art of retrovirus transgenic animals, specifically HIV transgenic animals, can be realized by following the teaching of the present invention.
Previously reported production of transgenic animals include:
(a) & (b) transgenic mice containing human globin genes, T. A. Stewart et al., Science, 217, 1046 (1982) and E. F. Wagner et al., Proc. Natl. Acad. Sci. USA, 78, 5016 (1981); PA0 (c) transgenic mice containing the human growth hormone gene fused to a metalothionein promoter sequence; R. D. Palmiter et al., Science, 222, 809 (1983); PA0 (d) transgenic mice with a recombinant gene containing the rat elastase I promoter and the codons for human growth hormone, D. M. Ornitz et al., Nature, 313, 600 (1985); PA0 (e) transgenic mice containing the human insulin gene, R. F. Selden and H. M. Howard, PCT patent application WO 87/07298, published Dec. 3, 1987; PA0 (f) several transgenic mice cited in a review by G. Skangos and C. Bieberich, Advances in Genetics, 24, 285 (1987); PA0 (g) several transgenic mice cited in a review by R. M. Strojek et al. entitled "The Use of Transgenic Animal Techniques for Livestock Improvement" in "Genetic Engineering: Principles and Methods", J. K. Setlow, Ed., Vol. 10, New York, N.Y., USA, 1988; PA0 (h) transgenic non-human animals having a transgene with an activated oncogene sequence, P. Leder and T. A. Stewart, U.S. Pat. No. 4,736,866, issued Apr. 12, 1988; PA0 (i) several transgenic animals cited in a review by J. Van Brunt, Bio/Technology, 6, 1149 (1988); PA0 (j) transgenic animals, containing hormone encoded genes, from which hormones can be harvested, R. M. Evans et al., U.S. Pat. No. 4,870,009, issued Sep. 26, 1989; PA0 (k) transgenic animals containing a gene capable of producing recombinant proteins in the animal's milk, H. Meade and N. Lonberg, U.S. Pat. No. 4,873,316, issued Oct. 10, 1989; and PA0 (l) transgenic mice, useful for analysis of hair growth, having recombinant genes comprising the regulatory elements involved in the expression of hair specific genes, A. R. McNab et al., Canadian patent application 2004156, published May 31, 1990. PA0 (a) a line of transgenic mice carrying only the long terminal repeat (LTR) of HIV fused to the chloramphenicol acetyl transferase (CAT) reporter gene, which were mated to transgenic mice of the opposite sex carrying the HIV-1 tat gene fused to a control element of the murine A.alpha. crystallin gene to give progeny carrying both genes; hence, only a small portion of the HIV-1 genome is involved, J. S. Khillan et al., Nucleic Acids Res., 16, 1423 (1988); PA0 (b) transgenic mice having the HIV-1 tat gene linked to the HIV-1 LTR in the absence of other parts of the HIV-1 genome and which developed skin lesions resembling Kaposi's sarcoma, J. Vogel et al., Nature, 335, 606 (1988); PA0 (c) a single founder line of transgenic mice carrying a complete HIV-1 proviral genome wherein the offspring developed a disease syndrome resembling some aspects of the symptoms of AIDS patients, and marked by the presence of HIV in the tissues of the offspring and premature death thereof, J. M. Leonard et al., Science, 242, 1665 (1988); PA0 (d) four lines of transgenic mice containing the HIV-1 LTR linked to the CAT reporter gene but no other parts of the HIV-1 genome and for which CAT activity was observed among mononuclear cells and maximally in Langerhans' cells, J. Leonard et al., AIDS Res. Hum. Retroviruses, 5, 421 (1989); PA0 (e) trangenic progeny from three founder mice bearing chromosomally integrated copies of partial HIV-1 proviral DNA (rendered non-infectious by deletion of the gag-pol sequences) and which developed glomerulosclerosis, P. Dickie et al., Sixth International Conference on AIDS, San Francisco, Vols 1-3, abstract Th.A. 290 (1990); PA0 (f) transgenic mice carrying HIV TAT and NEF genes under control of the HIV LTR, but no other parts of the HIV genome, as well as two transgenic founders carrying the HIV NEF gene construct (without TAT), J. Dinchuk et al., Sixth International Conference on AIDS, San Francisco, Vols. 1-3, abstract Th.A. 291 (1990); PA0 (g) transgenic mice bearing the LTR of HIV (but no other parts of the HIV-1 genome) fused to the reporter gene CAT, one line of which showed CAT activity in the cerebellum, E. Harlan and O. Prakash, Society for Neuroscience, Abstracts, Vol. 16, 353 (1990), abstract 154.1; and PA0 (h) three lines of transgenic mice carrying a partial HIV genome comprising the HIV-1 LTR fused to the simian virus 40 early region, for which the transgene was observed in the lymphoid tissue and skin of the mice, J. Skowronski, J. Virol., 65, 754 (1991). PA0 (a) isolating a fertilized oocyte from a first female animal; PA0 (b) transferring the transgene into the fertilized oocyte; PA0 (c) transferring the fertilized oocyte containing the transgene to the uterus of the same species as the first animal; PA0 (d) maintaining the second female animal such that PA0 (a) administering the test compound to the transgenic animal, and PA0 (b) examining the effect or significance of the test compound on the expressed gag proteins in the animal by monitoring the expression levels of the proteins. PA0 (a) administering the test compound to the transgenic animal, and PA0 (b) examining the effect or significance of the test compound on the expressed gag and envelope proteins and the gag protein cleavage products in the animal by monitering the expression levels thereof.
Previous reports of transgenic mice carrying all or part of the HIV-1 genome include:
The transgenic animal of the present application is distinguished from the transgenic animals of the prior art in that it carries a non-infectious transgene having the complete coding sequence of the HIV genome, the transgene being capable of producing high levels of HIV proteins in several tissues and certain body fluids of the animal. The attainment of the present transgenic mammal is surprising indeed in view of previous reports that expression of HIV proteins was found to be very low in mouse fibroblasts transfected with the HIV genome; for example, see J. A. Levy et al., Science, 232, 998 (1986).
The term "HIV" as used includes both type 1 and type 2 human immunodeficiency viruses and their strains, unless it is used within the context of a specific embodiment related to type 1 or type 2 virus. The terms "HIV-1" and "HIV-2" are used to distinguish the type 1 virus and its strains from the type 2 virus and its strains. The HIV-1 and HIV-2 genomes, and the DNA sequences of HIV-1 and HIV-2, and respective strains are described in the publication, "Human Retrovirus And AIDS 1991",Eds. G. Myer et al., Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Almos, N. Mex., 87545, USA. The latter publication hereby is incorporated by reference.